Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Exposure to IL-6 (10 ng/mL, 12 hours) induced increases in ODAM mRNA levels in Ca9-22 cells (A), HSY cells (B), and Sa3 cells (C), which were inhibited by SC144 (1 μM). ODAM and GAPDH mRNA levels were analyzed using real-time PCR. The experiments were performed in triplicate. Quantitative analyses of the data sets are presented with standard deviations. ODAM: odontogenic ameloblast-associated protein, IL-6: interleukin-6, GAPDH: glyceraldehyde 3-phosphate dehydrogenase, PCR: polymerase chain reaction. Significant differences from the control are indicated by ** P <0.01.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Control

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Protein levels were assessed using Western blotting following stimulation with IL-6 (10 ng/mL) for durations of 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours in Ca9-22 cells. Densitometric analyses of the Western blot results for ODAM, p-STAT3, STAT3, p-p65, p65, p-IKKα/β, IKKβ, and IKKα are presented. The intensity of the α-tubulin band was normalized to 1, and graphs were generated to illustrate the relative intensities of the other protein bands. IL-6: interleukin-6, ODAM: odontogenic ameloblast-associated protein, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, p65: nuclear factor-kappa B p65, p-p65: phosphorylated p65, IKKα: IκB kinase α, IKKβ: IκB kinase β, p-IKKα/β: phosphorylated IκB kinase α/β. The data represent the mean ± standard deviation from 3 independent experiments, with * P <0.05 and ** P <0.01 indicating statistical significance.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Western Blot, Generated, Standard Deviation

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: (A) LUC activity of −85ODAM, −116ODAM, −174ODAM, −200ODAM, −300ODAM, −330ODAM, −480ODAM, −700ODAM, and −950ODAM constructs were increased by IL-6 (10 ng/mL) after 12 hours of treatment in Ca9-22 cells. Transcriptional activity was determined from 4 separate transfections, and the combined values are expressed with SDs. (B) Effects of kinase inhibitors on the LUC activity of −480ODAM, stimulated by IL-6. Transient transfection analyses of −480ODAM in Ca9-22 cells, with or without IL-6 (10 ng/mL) treatment, were conducted alongside the effects of inhibitors of PKC (H7, 5 μM), PKA (KT5720, 100 nM), tyrosine kinase (HA, 1 μM), MEK1/2 (U0126, 5 μM), PI3K (LY294002, 10 μM), NF-κB (triptolide, 100 nM), STAT3 (stattic, 10 µM), STAT1 (fludarabine, 10 µM), and gp130 (SC144, 1 μM). Transcriptional activity was determined from 3 separate transfections, with the combined values expressed with SDs. ODAM: odontogenic ameloblast-associated protein, IL-6: interleukin-6, LUC: luciferase, SD: standard deviation, PKC: protein kinase C, PKA: protein kinase A, MEK1/2: mitogen-activated protein kinase kinase 1/2, PI3K: phosphatidylinositol 3-kinase, NF-κB: nuclear factor-kappa B, STAT: signal transducer and activator of transcription, gp130: glycoprotein 130. Significant differences from the control are indicated by ** P <0.01.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Activity Assay, Construct, Transfection, Luciferase, Standard Deviation, Control

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: Supershift gel shift assays were performed using anti-YY1, C/EBPβ, GATA, STAT3, and p-STAT3 antibodies for each reaction. The DNA-protein complexes were then separated by electrophoresis on a 6% polyacrylamide gel and visualized using the ChemiDoc Touch MP Imaging System. YY1: yin yang 1, C/EBP: CCAAT/enhancer-binding protein, GATA: GATA binding protein, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Gel Shift, Electrophoresis, Imaging, Binding Assay

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: (A) PCR bands corresponding to DNA-protein complexes immunoprecipitated with antibodies indicated that YY1, C/EBPβ, GATA, STAT3, and p-STAT3 interact with chromatin fragments containing the YY1, C/EBP, GATA, and GATE1-3 sequences. These interactions were increased in Ca9-22 cells following stimulation with IL-6 (10 ng/mL). Input DNA served as a control in the PCR analysis. (B) ChIP analyses were conducted to assess the binding of transcription factors to YY1, C/EBP, GATA, and GATE1-3 in the presence of IL-6 and various kinase inhibitors. Treatment with HA, U0126, LY294002, and triptolide almost completely abolished, and KT5720 partially inhibited, the IL-6-induced (10 ng/mL, 12 hours) binding of YY1, C/EBPβ, and GATA transcription factors to their respective DNA sequences. Additionally, treatment with stattic almost completely abolished, while fludarabine did not inhibit, the IL-6-induced binding of STAT3 transcription factors to GATE1-3. ChIP: chromatin immunoprecipitation, YY1: yin yang 1, C/EBP: CCAAT/enhancer-binding protein, GATA: GATA binding protein, GATE1-3: interferon-γ activated transcriptional element sites 1-3, ODAM: odontogenic ameloblast-associated protein, PCR: polymerase chain reaction, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, IL-6: interleukin-6.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Immunoprecipitation, Control, Binding Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction

Journal: Journal of Periodontal & Implant Science

Article Title: Interleukin-6 regulates human ODAM gene expression in gingival epithelial cells

doi: 10.5051/jpis.2402980149

Figure Lengend Snippet: IL-6–regulated ODAM gene expression is supported by protein–DNA interactions at the YY1, C/EBP, GATA, and GATE1-3. The signaling pathway involves gp130, JAK, STAT3, p-STAT3, PI3K, AKT, NF-κB, GPCR, SOCS3, SHP2, ERK, Grb2, Shc, Ras, and Raf. ODAM: odontogenic ameloblast-associated protein, IL-6: interleukin 6, YY1: yin yang 1, C/EBP: CCAAT/enhancer-binding protein, GATA: GATA binding protein, GATE1-3: interferon-γ activated transcriptional element 1-3, gp130: glycoprotein 130, STAT3: signal transducer and activator of transcription 3, p-STAT3: phosphorylated STAT3, PI3K: phosphatidylinositol 3-kinase, NF-κB: nuclear factor-kappa B, GPCR: G protein-coupled receptor.

Article Snippet: Twelve hours after transfection, the cells were switched to a serum-free medium for 12 hours and then treated with one of the following inhibitors for 30 minutes: 5 μM protein kinase C inhibitor H7 (Seikagaku Corporation, Tokyo, Japan), 100 nM protein kinase A inhibitor KT5720 (Sigma-Aldrich), 5 μM mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitor U0126 (Promega), 10 μM phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (Calbiochem, San Diego, CA, USA), 100 nM NF-κB inhibitor triptolide (Tocris Bioscience, Bristol, UK), 1 μM glycoprotein 130 (gp130) inhibitor SC144 (MedChemExpress, Monmouth Junction, NJ, USA), 10 μM STAT1 inhibitor fludarabine (NSC 118218; Selleck Chemicals, Houston, TX, USA), or 10 μM STAT3 inhibitor stattic (Axon Medchem, Reston, VA, USA).

Techniques: Gene Expression, Binding Assay

Journal: Cell reports

Article Title: A basophil-fibroblast pro-inflammatory axis fuels type 2 skin inflammation

doi: 10.1016/j.celrep.2025.116114

Figure Lengend Snippet: (A) Experimental outline of Il4ra fl/fl vs. Il4ra ΔPdgfra mice treated with vehicle or SC144 during MC903. (B) Representative IF staining of pSTAT3 (red) and PDGFRA (green) in Il4ra fl/fl and Il4ra ΔPdgfra with vehicle or SC144 in d7 MC903, quantified at right. * p < 0.05, ** p < 0.01, one-way ANOVA with Dunn’s multiple comparisons test. (C) Representative gross clinical images and H&E from Il4ra fl/fl vs. Il4ra ΔPdgfra mice with vehicle or SC144 at d7 MC903. (D) Skin thickness measurements of Il4ra fl/fl vs. Il4ra ΔPdgfra mice with vehicle or SC144 at d7 MC903. Data are mean ± S.E.M. * p < 0.05, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. (E) Skin thickness quantification of Il4ra fl/fl vs. Il4ra ΔPdgfra mice with vehicle or SC144 at d7 MC903. Data are mean ± S.E.M. * p < 0.05, *** p < 0.001, one-way ANOVA with Tukey’s multiple comparisons test. (F) CD45 + proportion in Il4ra fl/fl vs. Il4ra ΔPdgfra mice at d7 MC903. Data are mean ± S.E.M. * p < 0.05, ** p < 0.01, one-way ANOVA with Tukey’s multiple comparisons test. (G) Proposed model of basophil-induced OSM and IL-4/13 synergistic stimulation of pro-inflammatory fibroblasts that recruit immune cells to promote inflammation.

Article Snippet: SC144 gp130 inhibitor , MedChemExpress , HY-15614.

Techniques: Staining